Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Epigenetic reprogramming governs EcSOD expression during human mammary epithelial cell differentiation, tumorigenesis and metastasis

Oncogene volume 33, pages 358–368 (2014)Cite this article

Subjects

Abstract

Expression of the antioxidant enzyme EcSOD in normal human mammary epithelial cells was not recognized until recently. Although expression of EcSOD was not detectable in non-malignant human mammary epithelial cells (HMEC) cultured in conventional two-dimensional (2D) culture conditions, EcSOD protein expression was observed in normal human breast tissues, suggesting that the 2D-cultured condition induces a repressive status of EcSOD gene expression in HMEC. With the use of laminin-enriched extracellular matrix (lrECM), we were able to detect expression of EcSOD when HMEC formed polarized acinar structures in a 3D-culture condition. Repression of the EcSOD-gene expression was again seen when the HMEC acini were sub-cultured as a monolayer, implying that lrECM-induced acinar morphogenesis is essential in EcSOD-gene activation. We have further shown the involvement of DNA methylation in regulating EcSOD expression in HMEC under these cell culture conditions. EcSOD mRNA expression was strongly induced in the 2D-cultured HMEC after treatment with a DNA methyltransferase inhibitor. In addition, epigenetic analyses showed a decrease in the degree of CpG methylation in the EcSOD promoter in the 3D versus 2D-cultured HMEC. More importantly, >80% of clinical mammary adenocarcinoma samples showed significantly decreased EcSOD mRNA and protein expression levels compared with normal mammary tissues and there is an inverse correlation between the expression levels of EcSOD and the clinical stages of breast cancer. Combined bisulfite restriction analysis analysis of some of the tumors also revealed an association of DNA methylation with the loss of EcSOD expression in vivo. Furthermore, overexpression of EcSOD inhibited breast cancer metastasis in both the experimental lung metastasis model and the syngeneic mouse model. This study suggests that epigenetic silencing of EcSOD may contribute to mammary tumorigenesis and that restoring the extracellular superoxide scavenging activity could be an effective strategy for breast cancer treatment.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

References

  1. Oberley LW, Buettner GR . Role of superoxide dismutase in cancer: a review. Cancer Res 1979; 39: 1141–1149.

    CAS  Google Scholar 

  2. Oberley LW, Oberley TD, Buettner GR . Cell division in normal and transformed cells: the possible role of superoxide and hydrogen peroxide. Med Hypotheses 1981; 7: 21–42.

    Article  CAS  Google Scholar 

  3. Gius D, Spitz DR . Redox signaling in cancer biology. Antioxid Redox Signal 2006; 8: 1249–1252.

    Article  CAS  Google Scholar 

  4. Behrend L, Henderson G, Zwacka RM . Reactive oxygen species in oncogenic transformation. Biochem Soc Trans 2003; 31 (Pt 6): 1441–1444.

    Article  CAS  Google Scholar 

  5. Oberley LW . Mechanism of the tumor suppressive effect of MnSOD overexpression. Biomed Pharmacother 2005; 59: 143–148.

    Article  CAS  Google Scholar 

  6. Teoh-Fitzgerald ML, Fitzgerald MP, Jensen TJ, Futscher BW, Domann FE . Genetic and Epigenetic Inactivation of Extracellular Superoxide Dismutase Promotes an Invasive Phenotype in Human Lung Cancer by Disrupting ECM Homeostasis. Mol Cancer Res 2012; 10: 40–51.

    Article  CAS  Google Scholar 

  7. Teodoridis JM, Strathdee G, Brown R . Epigenetic silencing mediated by CpG island methylation: potential as a therapeutic target and as a biomarker. Drug Resist Updat 2004; 7: 267–278.

    Article  CAS  Google Scholar 

  8. Marklund SL . Extracellular superoxide dismutase in human tissues and human cell lines. J Clin Invest 1984; 74: 1398–1403.

    Article  CAS  Google Scholar 

  9. Teoh ML, Fitzgerald MP, Oberley LW, Domann FE . Overexpression of extracellular superoxide dismutase attenuates heparanase expression and inhibits breast carcinoma cell growth and invasion. Cancer Res 2009; 69: 6355–6363.

    Article  CAS  Google Scholar 

  10. Petersen OW, Ronnov-Jessen L, Howlett AR, Bissell MJ . Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells. Proc Natl Acad Sci USA 1992; 89: 9064–9068.

    Article  CAS  Google Scholar 

  11. Weaver VM, Lelievre S, Lakins JN, Chrenek MA, Jones JC, Giancotti F et al. Beta4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium. Cancer Cell 2002; 2: 205–216.

    Article  CAS  Google Scholar 

  12. Kenny PA, Lee GY, Myers CA, Neve RM, Semeiks JR, Spellman PT et al. The morphologies of breast cancer cell lines in three-dimensional assays correlate with their profiles of gene expression. Mol Oncol 2007; 1: 84–96.

    Article  CAS  Google Scholar 

  13. Edovitsky E, Elkin M, Zcharia E, Peretz T, Vlodavsky I . Heparanase gene silencing, tumor invasiveness, angiogenesis, and metastasis. J Natl Cancer Inst. 2004; 96: 1219–1230.

    Article  CAS  Google Scholar 

  14. Adachi T, Kodera T, Ohta H, Hayashi K, Hirano K . The heparin binding site of human extracellular-superoxide dismutase. Arch Biochem Biophys 1992; 297: 155–161.

    Article  CAS  Google Scholar 

  15. Hicks CL, Bucy J, Stofer W . Heat inactivation of superoxide dismutase in bovine milk. J Dairy Sci 1979; 62: 529–532.

    Article  CAS  Google Scholar 

  16. Hicks CL . Occurrence and consequence of superoxide dismutase in milk products: a review. J Dairy Sci 1980; 63: 1199–1204.

    Article  CAS  Google Scholar 

  17. Jones PA . DNA methylation and cancer. Oncogene 2002; 21: 5358–5360.

    Article  CAS  Google Scholar 

  18. Laukkanen MO, Mannermaa S, Hiltunen MO, Aittomaki S, Airenne K, Janne J et al. Local hypomethylation in atherosclerosis found in rabbit ec-sod gene. Arterioscler Thromb Vasc Biol 1999; 19: 2171–2178.

    Article  CAS  Google Scholar 

  19. Garbe JC, Holst CR, Bassett E, Tlsty T, Stampfer MR . Inactivation of p53 function in cultured human mammary epithelial cells turns the telomere-length dependent senescence barrier from agonescence into crisis. Cell Cycle 2007; 6: 1927–1936.

    Article  CAS  Google Scholar 

  20. Novak P, Jensen TJ, Garbe JC, Stampfer MR, Futscher BW . Stepwise DNA methylation changes are linked to escape from defined proliferation barriers and mammary epithelial cell immortalization. Cancer Res 2009; 69: 5251–5258.

    Article  CAS  Google Scholar 

  21. Li Y, Pan J, Li JL, Lee JH, Tunkey C, Saraf K et al. Transcriptional changes associated with breast cancer occur as normal human mammary epithelial cells overcome senescence barriers and become immortalized. Mol Cancer 2007; 6: 7.

    Article  Google Scholar 

  22. Chen LH, Bissell MJ . A novel regulatory mechanism for whey acidic protein gene expression. Cell Regul 1989; 1: 45–54.

    Article  CAS  Google Scholar 

  23. Jolivet G, Pantano T, Houdebine LM . Regulation by the extracellular matrix (ECM) of prolactin-induced alpha s1-casein gene expression in rabbit primary mammary cells: role of STAT5, C/EBP, and chromatin structure. J Cell Biochem 2005; 95: 313–327.

    Article  CAS  Google Scholar 

  24. Xu R, Spencer VA, Bissell MJ . Extracellular matrix-regulated gene expression requires cooperation of SWI/SNF and transcription factors. J Biol Chem 2007; 282: 14992–14999.

    Article  CAS  Google Scholar 

  25. Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, Brudno Y et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 2009; 324: 930–935.

    Article  CAS  Google Scholar 

  26. Valinluck V, Sowers LC . Endogenous cytosine damage products alter the site selectivity of human DNA maintenance methyltransferase DNMT1. Cancer Res 2007; 67: 946–950.

    Article  CAS  Google Scholar 

  27. Wu H, D'Alessio AC, Ito S, Xia K, Wang Z, Cui K et al. Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells. Nature 2011; 473: 389–393.

    Article  CAS  Google Scholar 

  28. Hewetson A, Hendrix EC, Mansharamani M, Lee VH, Chilton BS . Identification of the RUSH consensus-binding site by cyclic amplification and selection of targets: demonstration that RUSH mediates the ability of prolactin to augment progesterone-dependent gene expression. Mol Endocrinol 2002; 16: 2101–2112.

    Article  CAS  Google Scholar 

  29. Vukicevic S, Kleinman HK, Luyten FP, Roberts AB, Roche NS, Reddi AH . Identification of multiple active growth factors in basement membrane Matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components. Exp Cell Res 1992; 202: 1–8.

    Article  CAS  Google Scholar 

  30. Henrotin Y, Deberg M, Mathy-Hartert M, Deby-Dupont G . Biochemical biomarkers of oxidative collagen damage. Adv Clin Chem 2009; 49: 31–55.

    Article  CAS  Google Scholar 

  31. Bernfield M, Gotte M, Park PW, Reizes O, Fitzgerald ML, Lincecum J et al. Functions of cell surface heparan sulfate proteoglycans. Annu Rev Biochem 1999; 68: 729–777.

    Article  CAS  Google Scholar 

  32. Raats CJ, Bakker MA, van den Born J, Berden JH . Hydroxyl radicals depolymerize glomerular heparan sulfate in vitro and in experimental nephrotic syndrome. J Biol Chem 1997; 272: 26734–26741.

    Article  CAS  Google Scholar 

  33. Lee GY, Kenny PA, Lee EH, Bissell MJ . Three-dimensional culture models of normal and malignant breast epithelial cells. Nat Methods 2007; 4: 359–365.

    Article  CAS  Google Scholar 

  34. Tang X, Sun Z, Runne C, Madsen J, Domann F, Henry M et al. A critical role of Gbetagamma in tumorigenesis and metastasis of breast cancer. J Biol Chem 2011; 286: 13244–13254.

    Article  CAS  Google Scholar 

  35. Blaschke RJ, Howlett AR, Desprez PY, Petersen OW, Bissell MJ . Cell differentiation by extracellular matrix components. Methods Enzymol 1994; 245: 535–556.

    Article  CAS  Google Scholar 

  36. Case AJ, McGill JL, Tygrett LT, Shirasawa T, Spitz DR, Waldschmidt TJ et al. Elevated mitochondrial superoxide disrupts normal T cell development, impairing adaptive immune responses to an influenza challenge. Free Radic Biol Med 2011; 50: 448–458.

    Article  CAS  Google Scholar 

  37. Teoh ML, Sun W, Smith BJ, Oberley LW, Cullen JJ . Modulation of reactive oxygen species in pancreatic cancer. Clin Cancer Res 2007; 13: 7441–7450.

    Article  CAS  Google Scholar 

  38. Shan W, Zhong W, Zhao R, Oberley TD . Thioredoxin 1 as a subcellular biomarker of redox imbalance in human prostate cancer progression. Free Radic Biol Med 2010; 49: 2078–2087.

    Article  CAS  Google Scholar 

  39. Rose SL, Fitzgerald MP, White NO, Hitchler MJ, Futscher BW, De Geest K et al. Epigenetic regulation of maspin expression in human ovarian carcinoma cells. Gynecol Oncol 2006; 102: 319–324.

    Article  CAS  Google Scholar 

  40. Svensson RU, Barnes JM, Rokhlin OW, Cohen MB, Henry MD . Chemotherapeutic agents up-regulate the cytomegalovirus promoter: implications for bioluminescence imaging of tumor response to therapy. Cancer Res 2007; 67: 10445–10454.

    Article  CAS  Google Scholar 

  41. Drake JM, Gabriel CL, Henry MD . Assessing tumor growth and distribution in a model of prostate cancer metastasis using bioluminescence imaging. Clin Exp Metastasis 2005; 22: 674–684.

    Article  Google Scholar 

Download references

Acknowledgements

The authors thank Dr James Crapo (National Jewish Medical and Research Center, Denver, CO, USA) for providing antibodies to EcSOD and Dr Michael Henry (Department of Molecular Physiology and Biophysics, University of Iowa, IA, USA) for providing the 4T1.luc cells. We also thank Justin Fishbaugh from the Flow Cytometry Core Facility for assisting with the DCFH assays as well as the Central Microscopy Research Facility. This study was supported by NIH grant R01 CA073612 and R01 CA115438 (FE Domann), Susan G Komen for the Cure grant KG080437 (M Teoh-Fitzgerald), SFRBM Research Mini-Fellowship (M Teoh-Fitzgerald), Oberley Seed Grant (M Teoh-Fitzgerald), and Carver Research Program of Excellence in Redox Biology and Medicine, and by the Holden Comprehensive Cancer Center Breast Cancer Research Fund.

Author information

Authors and Affiliations

  1. Department of Radiation Oncology, Free Radical and Radiation Biology Program, University of Iowa, Iowa City, IA, USA

    M L Teoh-Fitzgerald, M P Fitzgerald & F E Domann

  2. Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, WI, USA

    W Zhong

  3. Department of Pathology, University of Iowa, Iowa City, IA, USA

    R W Askeland

Authors
  1. M L Teoh-Fitzgerald
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M P Fitzgerald
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. W Zhong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. R W Askeland
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. F E Domann
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to M L Teoh-Fitzgerald or F E Domann.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Teoh-Fitzgerald, M., Fitzgerald, M., Zhong, W. et al. Epigenetic reprogramming governs EcSOD expression during human mammary epithelial cell differentiation, tumorigenesis and metastasis. Oncogene 33, 358–368 (2014). https://doi.org/10.1038/onc.2012.582

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2012.582

Keywords

This article is cited by

Search

Advanced search

Quick links